Sarcopenia is the progressive loss of skeletal muscle mass during aging leading factor that contributes to frailty, debilitating injuries, loss of independence, and reduced quality of life in the elderly. Unfortunately, sarcopenia progresses despite interventions such as increased physical activity and improved diet. During sarcopenia the age-related decreases in muscle strength result from a combination of loss of muscle mass (atrophy) and reduced muscle specific force (i.e., muscle force per unit of cross-sectional area). Increasingly, it is thought that the muscle weakness that accompanies sarcopenia, not the loss of muscle size per se, is the principal contributor to disability. We have recently found that elements of contractile force generation in skeletal muscle are dependent on store-operated Ca2+ entry (SOCE) and that this capacity is lost in aging skeletal muscle. This altered SOCE is directly associated with reduced content of mitsugumin 29 (MG29), a muscle-specific protein belonging to the synaptophysin (SYPL2) family containing a MARVEL domain involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the proper formation of the transverse-tubule (TT) system and efficient SOCE. This leads to a link between reduced MG29 expression in aged muscle and the development of muscle dysfunction in sarcopenia. Skeletal muscles from young mg29-/- mice are similar to that from aged wild-type (WT) mice in that they demonstrate decreased specific contractile force, reduced SOCE, reduced Ca release from the SR, reduced content of MG29, and altered morphology (i.e., increased missing triad junctions and formation of SR aggregates). Therefore, our data demonstrate that key aspects of skeletal muscle aging are present in mg29-/- mice, making this model applicable for this line of research. In this proposal we specifically hypothesize that MG29 is required for proper TT formation and SOCE function in skeletal muscle through the action of specific protein domains. Decreased MG29 content in normal aged muscle leads to defective SOCE, which results in decreased availability of Ca2+ for contractility and age-related loss of muscle strength not accounted for by muscle atrophy. We will test this hypothesis using two specific aims that will both examine the mechanistic basis for MG29 function in skeletal muscle and also provide translational value for the treatment of sarcopenia. Aim 1 will elucidate the contribution of MG29 to SOCE, SR Ca release and contractility in aged skeletal muscle by using electroporation of MG29 cDNA or RNAi constructs to alter the expression of MG29 and concomitantly evaluate SOCE function, SR Ca release, and muscle specific contractile force. Additional studies in Aim 1 will use viral gene delivery to modulate MG29 expression in mouse muscle and establish the therapeutic value of this approach. In Aim 2 we will establish the molecular mechanisms of MG29 regulation of SOCE in skeletal muscle and how these are altered in aging using molecular biology and biochemical approaches to resolve the protein motifs and specific residues responsible for the mechanism of action of MG29 in muscle fibers.